This application is a 371 of PCT/JP98/02389, filed May 29, 1998.
The present invention relates to a method for stably retaining an extremely minute amount of a solution used for a chemical reaction or the like on a surface of a glass plate or the like, a method for effecting a reaction such as PCR using minute amount of reagents, and a reaction vessel suitable for use therein.
In recent years, reactions such as PCR (polymerase chain reaction) and EIA (enzyme immunoassay) have been utilized in the field of biotechnology. In these reactions, reduction of an amount of a reaction solution to a minute amount is important from the viewpoint of cost reduction or the like. Ink jet technique which deposits ink on paper to visualize letters, characters and images was originally developed for printing. However, ink jet technique is capable of precisely dispencing a minute amount of a liquid, and thus applications thereof have been attempted as a technique for reducing an amount of a reaction solution to a minute amount.
U.S. Pat. No. 4,877,745 and BioTechniques 15, 324 (1993) disclose a method for utilizing ink jet technique as a dispencer is disclosed. U.S. Pat. No. 5,449,754 and U.S. Pat. No. 5,474,796 disclose application of ink jet technique to organic chemical syntheses. Japanese Unexamined Patent Publication No. 262256/1992, Japanese Unexamined Patent Publication No. 289457/1992 and Analytical Chemistry 67, 3051 disclose application of ink jet technique to immunological reactions. For example, Japanese Unexamined Patent Publication No. 262256/1992 discloses a method which comprises preliminary printing a necessary reagent on a film by ink-jetting, adding a sample solution thereto in an amount on the order of 50 xcexcl (microliter) at the time of use to permit the reagent to dissolve in the solution and to thereby effect reaction, followed by washing and color development to perform detection.
As described above, it has been attempted to utilize ink-jetting as a dispenser for a reaction solution in a chemical reaction. In this connection, when a number of reactions are effected in parallel in a clinical test or the like, it is desired to minimize an amount of each individual droplet, for example, to reduce the amount to 100 nl (nanoliter) or less. However, no cases have been reported where a reaction in a clinical test, for example, PCR or an immunological reaction is effected in an extremely minute amount of a solution of the order of 10 nl to 1 pl (picoliter) by using ink-jetting. In the case of the above-mentioned Japanese Unexamined Patent Publication No. 262256/1992, the amount of the solution in the reaction is 50 xcexcl. This is not an extremely minute amount.
One of the reasons why no cases have been reported where a reaction in an extremely minute amount is effected using ink-jetting is that an extremely minute amount of a solution dispenced by ink-jetting vaporizes in about several seconds the in air. As in U.S. Pat. No. 5,449,754, with respect to an organic chemical reaction which completes in a moment, examples of a reaction in an extremely minute amount have been reported. However, a reaction in a clinical test generally takes a time period of several minutes to several hours. It is difficult to retain a reaction solution during the period in the air without evaporation of the reaction solution.
Further, when it is detected by fluorometry, colorimetry or the like whether a reaction occurred or not, the detection is difficult in an extremely minute amount of a reaction solution because a length of an optical path in the extremely minute amount of the-reaction solution is markedly shorter, as compared with a case of an ordinary amount of a reaction solution. Accordingly, a contrivance to elongate the length of the optical Ad path at the time of measurement is desired, although this is not indispensable for effecting a reaction in an extremely minute amount.
The present invention has been made in view of the above-described problems in the conventional techniques. It is, therefore, an object of the present invention to provide a method for stably retaining an extremely minute amount of a solution projected by, for example, ink jet method for a chemical reaction or the like for a long period of time without evaporation of the solution.
It is also an object of the present invention to provide a method for elongating a length of an optical path in order to facilitate optical detection with respect to an extremely minute amount of a solution projected by ink jet method or the like.
It is further an object of the present invention to provide a method for effecting a reaction, such as PCR, using a minute amount of a reagent projected by ink jet method, and a reaction vessel suitably used therein.
In the present invention, the above object is attained in such a manner that a layer of a liquid, such as an oil, which is hardly miscible with an extremely minute amount of a solution (minute droplet) intended to be retained is formed on a substrate such as a glass plate to retain the minute droplet in the liquid layer with the minute droplet in contact with a surface of the substrate. When the minute droplet is aqueous, the liquid layer applied onto the surface of the substrate may be oily. The minute droplet may be shot into the liquid layer applied onto the substrate from the surface of the liquid layer by ink jet technique or the like.
By appropriately selecting viscosity and thickness of the liquid layer formed on the substrate, the minute droplet shot from the surface of the liquid layer is permitted to deposit on the surface of the substrate and stably retained under cover of the liquid layer. The minute droplet is surrounded by the liquid which is substantially immiscible therewith, and thus evaporation thereof can greatly be reduced. Further, since the minute droplet is deposited and fixedly retained on the surface of the substrate, a reagent may further be added to the minute droplet as such by ink jet technique.
For example, when the minute droplet is shot into the liquid layer in an amount of 40 pl by ink jet method, optimum viscosity of the liquid layer is 20 to 50 cp and optimum thickness of the liquid layer is 20 to 30 xcexcm. If the liquid layer applied onto the surface of the substrate has too small a thickness, the minute droplet partially comes out of the liquid layer and evaporates. On the other hand, if the liquid layer has too large a thickness, the minute droplet is suspended in the liquid layer and positionally unsettled. Accordingly, addition of a reagent thereto or the like is difficult.
The surface of the substrate may have water repellency. The water repellency used herein is expressed as an amount of a rising angle (contact angle) of the droplet, which is deposited on the surface of the substrate, relative to the surface of the substrate. The larger the contact angle, the more water repellent. In the present invention, a surface of a substrate which has a larger contact angle of a droplet than that of a droplet deposited on a commonly used glass substrate is represented as having water repellency. The water repellency may be imparted to the substrate by preparing the substrate itself from a material having water repellecy, such as a polypropylene, or by coating a water repellent material over the substrate.
If the surface of the substrate has water repellency, an aqueous minute droplet deposited on the surface of the substrate has a smaller area of contact with the substrate and thus rises in the thickness direction of the liquid layer. Accordingly, when a chemical reaction which occurs in the minute droplet is detected by an optical method such as fluorometry, colorimetry or the like in the direction perpendicular to the substrate, the detection is facilitated because of the elongation of the length of the optical path. In this case, the substrate having water repellency or water repellent coating required not to affect the optical measurement of the minute droplet.
If the minute droplet is shot into the liquid layer covering the substrate to retain the minute droplet on the surface of the substrate and thereafter a transparent covering such as a slide glass is placed on the liquid layer, disturbance of the surface of the liquid layer due to convection caused under heating is prevented. The minute droplet is thereby prevented from coming out of the liquid layer and evaporating. Prior to the placement of the covering, the liquid may be added to the liquid layer.
It also is effective in stably retaining the minute droplet to place another aqueous solution in the liquid layer in the vicinity of the minute droplet shot in the liquid layer. The minute droplet retained in the liquid layer tends to dissolve into the surrounding liquid, for example, when heated in the reaction process, thereby undergoing decrease in volume. If another solution is placed in the vicinity of the aqueous minute droplet to be reacted, water content of the liquid layer can locally be increased in the vicinity of the minute droplet to provide an effect of preventing dissolution of the minute droplet to be reacted. By close placing minute droplets to be reacted instead of the placement of the dissolution-preventive aqueous solution, substantially the same dissolution reducing effect can be obtained.
From the viewpoint of elongation of a length of an optical path for facilitating optical measurement, it is desired to bring the minute droplet into contact also with the transparent covering placed on the liquid layer. This is realized by narrowing the space between the substrate and the covering to bring the minute droplet into contact with the covering, and then re-widenig the space between the substrate and the covering. For this purpose, it is preferred to use an elastic material as a spacer between the substrate and the covering. The action to narrow the space between the substrate and the covering can be effected, most simply, by pressing the covering with a finger. However, it may be effected by pressing the covering toward the substrate by means of a precise motor or a piezoelectric element. In this manner, by retaining the minute droplet in contact with both the upper surface of the substrate and the lower surface of the covering, a length of an optical path in the minute droplet in the direction perpendicular to the substrate can be elongated, and at the same time, it is possible to attain a regularlized optical path length. By virtue of this, precision and reliability of the measurement can be improved.
The reaction vessel according to the present invention comprises a transparent lower plate, a spacer having a thickness of 0.05 mm or less and a transparent upper plate, and the reaction vessel contains a solution in a space surrounded by the spacer.
The spacer may be made of a pressure sensitive adhesive double coated tape. It is preferred that a site of contact with the solution have a bovine serum alubmin coating.
With respect to a PCR chamber, some small-sized reaction chambers for PCR which are prepared by etching technique have been disclosed (see, for example, xe2x80x9cKikai-Ken Newsxe2x80x9d vol. 533, No. 5, pp. 6-8, 1996, published by Mechanical Engineering Research Laboratory of Agency of Industrial Science and Technology, written by Sohei Matsumoto). Although these reaction vessels are used with its inside entirely filled with a reaction solution for PCR and thus different from the reaction vessel of the present invention in manner of use, the reaction vessel of the present invention as shown in FIG. 21 has the following advantages as compared with the PCR chambers prepared by etching. {circle around (1)} The reaction vessel of the present invention has a simple structure and is inexpensive. This is because simple coating is applied to a mass-produced cover glass and the spacer is prepared by simple paper craft-like processing. {circle around (2)} The PCR chambers prepared by etching have rough finished surfaces, and thus nuclei of boil-bubbling are present therein. Due to this, bubbles are likely to develop, and temperature control is affected. (see the above-mentioned reference). On the other hand, the reaction vessel of the present invention is constructed by sticking materials made of ordinary glass or the like together. Accordingly, precise surface finishing can be effected on an as needed basis. {circle around (3)} In the PCR chambers prepared by etching, a coating method is employed in which, after completion of assembly of the whole, a chemical agent for coating is passed through fine flow paths and then dried to prevent clogging of the fine flow paths. However, coating treatments which can be carried out by this method are restricted to silicone coating and so forth. On the other hand, in the reaction vessel of the present invention, the coatings are performed prior to the assembly. Accordingly, any techniques such as utilization of a spinner and lamination of a polymer may be employed, and thus coating of the inner surface is easy.
The present invention can be applied to a reaction of a minute amount of a sample, for example, in a PCR method or immunoassay. Further, the present invention is effective for a biochemical reaction other than PCR, which involves a high temperature reaction (50xc2x0 C. or more), for example, LCR (ligase chain reaction) hybridization or the like. The reacting method according to the present invention comprises:
retaining a minute droplet in a layer of a liquid coated over a surface of a substrate with the minute droplet in contact with the surface of said substrate, said minute droplet and said liquid separating into two phases,
covering the surface of said liquid layer with a covering, and
effecting a reaction in said minute droplet. The minute droplet may be aqueous one containing DNA, and the liquid layer applied onto the surface of the substrate may be oil-based one.
The minute droplet can be deposited on the surface of the substrate by projecting it by ink jet technique. The minute droplet may be shot to a substrate preliminarily coated with an oily liquid layer by an ink jet head from the surface of the liquid layer so as to reach the surface of the substrate. The minute droplet may be shot directly to a surface of a substrate uncoated with a liquid layer to deposit thereon, and then an oily liquid layer is applied thereto.
It is desirable that the site of contact with the minute droplet be provided with an enzyme adsorption preventing agent, for example, a silicone coating. Further, it is preferable that the site of contact with the minute droplet be provided with a bovine serum albumin coating. In particular, the bovine serum albumin coating has an effect of improving deposition of the minute droplet, when the minute droplet is projected from an ink jet head to deposit on a surface of a substrate uncoated with a liquid layer.
In addition to these coatings, a various types of coatings Am, may be used as the inner coating of the reaction vessel. It is desired that the inner coating of the reaction vessel show {circle around (1)} few adsorption of enzyme and {circle around (2)} good deposition of an aqueous droplet. Coatings which satisfy these requirements include coatings of hydrophilic polymers. These coatings were originally developed to prevent adsorption of a protein in a medical appliance such as an artificial organ, and a capillary column. There have been many examples of the coatings, and the coatings can easily be applied to coating of the reaction vessel
As the hydrophilic polymer, for example, a polyvinyl alcohol, a polyethylene glycol, and a polyvinyl pyrrolidone may be mentioned. As the hydrophilic polymer which is water-insoluble and used for coating of a medical appliance or the like, for example, a poly(2-hydroxyethyl methacrylate), and a poly(2-methacryloyl oxyethyl phosphoryl choline-n-butyl methacrylate) copolymer may be mentioned (Hyomen Gijutsu, vol. 46, No. 10, pp. 880-886, 1995, Iwasaki et al.). Of these, a poly(2-hydroxyethyl methacrylate) which is on the market and readily available is taken by way of example to describe one mode of the coating.
0.2 g of a poly(2-hydroxyethyl methacrylate) (produced by Sigma Aldrich Japan Co.) was dissolved in 2 ml of dimethylhormamide (produced by Nacalai Tesque Co.). As shown in FIG. 41, the thus obtained coating liquid 143 was applied in an amount of 20 xcexcl onto a glass-exposed center portion of a cover glass 141 with a xe2x96xa1-shaped spacer seal 142 stuck thereon. In this connection, if the spacer seal 142 had not been provided, the applied liquid would be repelled by the surface of the glass and thus could not be applied uniformly. After completion of the application, the coating liquid was vacuum-dried to obtain a uniform transparent coating (as shown in FIG. 42).
To prevent adsorption of a protein on a surface of a material, in the field of capillary electrophoresis, methods have been known which comprises covalently fixing a polymer to inner surfaces of capillaries (made of fused quartz). These methods can be applied to coating of the reaction vessel. For example, fixation of a polyethylene glycol (Journal of Chromatography, vol. 471, pp. 429-436 (1989), G. J. M. BRUIN et al.), and fixation of a polyacrylamide (Journal of Chromatography, vol. 374, pp. 191-198 (1985), S. HJERTEN et al.) may be mentioned.
In addition, as a coating method having an effect comparable to that of the silicone coating, a fluororesin coating may be mentioned. Many methods for coating with a fluororesin have been known. In these cases, further treatment to promote deposition of the aqueous droplet, for example, application of bovine serum albumin should be effected.
Some surface treatments with chemicals entail high cost and show poor effect. In such cases, instead thereof, a method may be used which comprises sticking a resin thin film hardly susceptible to adsorption of a protein to the reaction vessel. As an example of the resin film which can be used, a fluororesin film xe2x80x9cCYTOPxe2x80x9d (produced by Asahi Glass Co., Ltd.) may be mentioned. In this case, because of high water repellency of the film surface, further treatment to promote deposition of the aqueous droplet, for example, application of bovine serum albumin should be effected. As a method for improving deposition of the aqueous droplet, irradiation of an ion beam or the like may be used (see, for example, Kobunshi Kako, vol. 44, No. 10, pp. 434-439, 1995, Sasabe et al.).
It is preferred that the liquid layer applied onto the surface of the substrate have a thickness of 100 xcexcm or less from the viewpoint of prevention of dissolution of the reagent solution in the oil.
According to the reacting method of the present invention, reduction of an amount of a solution per reaction batch to a minute amount is realized and thus cost of the reagent can be reduced. Further, with the same copy number, an amount of a reaction solution becomes smaller, a greater reduction of number of cycles is realized. For example, if the amount of the reaction solution is reduced to one-thousandth, log2 1000=10 cycles can be reduced theoretically. By the reduction of the number of cycles, reaction time is shortened, and side reactions (non-specific amplification, formation of dimers of a primer) are reduced. Accordingly, target detection limit is improved.
Further, according to the reacting method of the present invention, it is possible to assay PCR by quantization. Specifically, when a reaction solution is divided into a large number of batches, such a condition is obtained that one copy of a target or no target is present in each of the batches. When PCR is performed with the condition, number of batches where PCR progressed increases in proportion to a concentration of the target. Accordingly, by counting the number of the batches where PCR progressed, it is possible to precisely determine the concentration of the target in the original sample.